Human monoclonal antibody having fat-reducing effect

ABSTRACT

A purified polypeptide having an amino acid sequence that is substantial identical with the amino acid sequence of SEQ ID NO:1, SEQ ID NO:3, or both, in which the polypeptide binds low density lipoproteins (LDL) or oxidized LDL (oxLDL), or both, and particularly binds well to LDL cholesterol and oxidized LDL cholesterol. The polypeptide may be used in combination with conventional adjuvants or carrier substances for producing a drug having a fat-reducing effect and for producing drugs for aiding in the treatment of renal diseases.

This application is a U.S. national phase of International applicationno.

PCT/DE2004/002503, filed Nov. 12, 2004, and claims the benefit ofpriority of German application no. 103 53 175.0, filed Nov. 14, 2003.

The invention relates to a purified polypeptide (SAM-6.10) and its usein combination with conventional adjuvants and/or carrier substances forproducing a drug having a fat-reducing effect and for producing a drugfor treating renal diseases.

BACKGROUND OF THE INVENTION

Excessive cholesterol (hyperlipoproteinemia) in the body leads tosclerosis (arteriosclerotic plaque) of the interior layers of the bloodvessels, and to a gradual hardening and thickening of the arterialwalls. In an extreme case, it threatens blocking of the vessels, or inthe event of rupture of the plaque, thrombus formation. Arteriosclerosiswith its secondary illnesses (coronary illnesses, heart attacks,peripheral arterial diseases and strokes) is still the most frequentcause of death in the western world. Well over half of all the financialresources available for medical care are estimated to be spent for theconsequences of arteriosclerosis. To clarify the causes ofarteriosclerosis, various theories have been developed, of which thelipid theory is the most highly regarded.

In general, it can be stated that the higher the LDL cholesterol levelor level of oxidized LDL cholesterol in the blood, the high the risk ofvascular hardening, for example with the consequence of a heart attack.Overweight and hypercholesterolemia are the most important risk factorsfor the development of arteriosclerosis.

DEFINITIONS AND TERMS

Fats such as cholesterol are soluble neither in water nor blood plasma.To allow them nevertheless to be transported to individual body regions,the fats are bound to particular albuminous substances (proteins) assoon as they occur in the blood. These compounds, comprising lipids(fats) and proteins, are termed lipoproteins.

The “lipoproteins” of the plasma are high-molecular weight water-solublecomplex comprising lipids (cholesterol, triglycerides and phospholipids)and apolipoproteins. The cholesterol-containing lipoprotein LDLcholesterol causes arteriosclerosis and is also known as “bad”cholesterol, with the oxidized form of cholesterol being even moredangerous for the body.

“Cholesterol” is synthesized ubiquitously in the body and is anessential component of cell membranes and lipoproteins. In contrast tothe triglycerides and phospholipids, which are also synthesizedendogenously, the sterol ring of the cholesterol molecule can not bebroken down again; cholesterol is transformed into bile acid in theliver or excreted unchanged with the bile into the gut.

In plasma, cholesterol is present as 25 to 40% free (non-esterified)cholesterol and 60 to 75% esterified with unsaturated fatty acids. Bothforms together are known as the total cholesterol. Because of the lowwater solubility, cholesterol is transported in plasma as a complex withapolipoproteins. In the blood, about 70 percent of the total cholesterolis transported via low-density lipoproteins (LDL).

“Triglycerides” are esters of glycerine with three fatty acid residues.In the same way as cholesterol, the triglycerides are also transportedin plasma bound to apolipoproteins because of their poor solubility

“Lipoproteins” are synthesized in the liver or gut and transportliposoluble substances such as cholesterol in the blood.

Lipoproteins are classified according to their density into five densityclasses: chylomicrons, very low density lipoproteins (VDL), low densitylipoproteins (LDL) and high density lipoproteins (HDL). Chylomicrons,whose physiological concentration in fasting serum, unlike that of otherlipoproteins, is very low, are vehicles for transporting exogenousglycerides. The physiological distribution of the other lipoproteins isas follows: VLDL 10%, LDL 70% and HDL 20%. VLDL are the precursors ofLDL and vehicles for the transport of endogenous glycerides. LDL areproduced by hydrolysis of VLDL. LDL and HDL are both regulators ofcellular cholesterol homeostasis, with HDL also regulating lipolysis(splitting of triglycerides into glycerol and free fatty acids). LDLhave a diameter of about 20 nm. HDL are the smallest (7-10 nm) and themost protein-rich lipoproteins. Besides native LDL (LDL), oxidized LDL(oxLDL) is also detectable in the blood serum. oxLDL interacts withendogenous plasma proteins, particularly glycoproteins via specificligands and forms oxLDL glycoprotein complexes.

“Apolipoproteins” are a component of lipoproteins and, together withpolar lipids, as a kind of outer shell, surround the lipoprotein core,which is made up of hydrophobic lipids. With the exception of LDL, whichonly contains apoprotein B, the individual lipoprotein classes have aplurality of apolipoprotein classes with different structures.

Lipoprotein Transport

Cholesterol is principally transported via the two lipoprotein classesLDL and HDL. LDL are primarily responsible for cholesterol transport toperipheral cells that possess specific receptors for LDL. HDL permit andaccelerate the removal of cholesterol from the extra-hepatic cells andvessel walls and transfer it to the livers.

Pathology

As regards the pathology of lipid metabolism disorders, it can be saidin general that elevations in LDL cholesterol in conjunction withreductions in HDL cholesterol pose the most prominent increased risk ofarteriosclerosis. In the pathogeny, LDL, whose particles make asubstantial contribution to the formation of atherosclerotic plaques,and HDL therefore play contradictory roles. The quotients of totalcholesterol/HDL cholesterol and, in particular, LDL cholesterol/HDLcholesterol are the deciding factors for assessing the risk of heartattack. (Epidemiological studies (Framingham study) also refer to theprotective effect of HDL cholesterol.) The secondary diseases ofarteriorsclerosis, besides coronary heart disease and peripheralarterial diseases, also include heart and brain infarcts (strokes).

oxLDL, just like LDL, probably causes arteriosclerotic plaques, withoxLDL posing the biggest danger for the body.

But oxLDL also appears to play an important role in other diseases. Inpatients with chronic kidney failure and diabetes, the concentration ofoxLDL glycoprotein complexes is higher than in healthy patients.

LDL is also removed from the blood circulation by the liver and bymacrophages. Macrophages are cells of the immune system, which arecapable of phagocytosis of larger particles.

The “scavenger pathway” is a known model for explaining how cells engulfparticles (phagocytosis). The incorporation of solid particles (debris,foreign bodies, bacteria or LDL plaques) into the cell interior ofphagocytes, with subsequent intracellular breakdown, is carried out byphagocytosis. The phagocytic cells are known as phagocytes and consistpredominantly of tissue macrophages and mobile blood monocytes.

In phagocytosis, after the particles become attached to the cellmembranes of the phagocytes by binding to Fc and complement receptors onthe membrane, contractile structures are activated within thecytoplasma. Local inversions of the cell membranes then cause inclusionof the particles in the cytoplasmic vacuoles.

So-called scavenger phagocytes are found in lymph nodes in and along thefibre strands that comprise the medulla.

Whereas the lymph passage from the afferent to the efferent end of thelymph node, particular antigens are removed by the phagocytic cells.

It is further known that the adherence to phagocytic cells, such aspolymorphonuclear leucocytes and macrophages, is increased by theattachment of immunoglobulins (Ig) to the surface of bacteria (and otherantigens). It is supposed that the increased adherence is effected byattachment of the Fc component of the immunoglobulin to the Fc receptorsof the phagocytes. After the antigens have been made “appetizing” by theattachment of (or binding of) antibodies, the complex of antigen andantibodies is taken up and ingested faster by the phagocytic cells. Thecoating of the antibody surface with immunoglobulins is also known asopsonin-mediated (Fc) adherence, and plays an important role in theimmune response.

The antibodies binding to the surface of bacterial cells are capable offixing particular components of the extracellular liquids. In genericterms, these components are termed the “complement.” Animal tests haveshow that the phagocytosis of cells coated with antibodies is delayed inanimals lacking complement. It is thus obvious that opsonizationinvolves a synergy between antibodies and complement.

DESCRIPTION OF THE INVENTION

Drugs of the prior art for reducing LDL cholesterol act by inhibitingthe key enzyme for cholesterol synthesis (CSE). For example, onecholesterol synthesis inhibitor is a substance that has become knownunder the trade name Lipobay. The side effects of CSE inhibitors aregenerally considerable, including, inter alia, gastrointestinaldiseases, sleep disturbances, dizziness, visual disturbances, allergicreactions and hair loss. An approach that is only in the test stage,specifically only in the case of severe familial hypercholesterolemia,is somatic gene therapy, which consists of transferring the gene for theLDL receptor to autogenic liver cells.

So far, no substance that acts as a fat reducer and is largely free ofside effects has been available on the market. In particular, noantibodies that induce a severe intracellular accumulation oflipoproteins are so far known. Although the harmful role of lipoproteinsin renal diseases (lipid-induced damage of cells of the glomerulusfiltration system of the kidney) is known, there is so far noantibody-based therapy of renal diseases, in particularglomerulonecrosis.

The object of the invention consists in the generation of a newsubstance or of a new substance class for the preparation of a drug forreducing the LDL cholesterol or of the oxLDL cholesterol in humans andanimals with the advantageous goal of reducing the risk of infarct.

To achieve this object, a polypeptide is proposed, the amino acidsequence of which is essentially identical to the amino add sequence ofSEQ ID NO:1 and/or SEQ ID NO:3, and

-   -   binds the low density lipoproteins (LDL) and/or oxidized LDL        (oxLDL), in particular LDL cholesterol and/or oxidized LDL        cholesterol (oxLDL cholesterol),

The gist of the invention consists in the surprising observation that apurified polypeptide whose sequence corresponds entirely or partly tothe light (V_(L)) or heavy chain (V_(H)) of a human monoclonal antibody(SAM 6.10), effects reduction of the low density lipoprotein (LDL)and/or oxLDL. The discovery of this property, which suggests the use ofthe polypeptide as a fat reducer in a corresponding pharmaceuticalformulation, was made in the course of biochemical characterization ofthe polypeptide. Advantageously, the binding of the polypeptideaccording to the invention or of the fragments of the polypeptide to LDLand/or oxLDL and to VLDL, the precursors of LDL, is stronger than thebinding to HDL. As a result of this property, the polypeptide accordingto the invention leads to a low value for the respective quotient ofLDL/HDL and/or oxLDL/HDL, and thus minimizes the risk of infarct.

The specific binding of the polypeptide to low density lipoproteins (LDLand/or oxLDL), or LDL cholesterol-and/or oxLDL cholesterol has beenproven experimentally by the ELISA method. In the same experiment, itwas also possible to prove that the binding of the substance accordingto the invention to high density lipoproteins (HDL) is weak.

The antibody according to the invention comprises the groups termed VL,VH, FV, FC, Fab, Fab′, F(ab′)₂ according to the conventionalnomenclature for describing antibodies. The aforementioned groups arealso known as fragments. It is entirely possible that an individualfragment is the cause of the fat-reducing effect of the polypeptideaccording to the invention. In a further development of the substanceaccording to the invention, the substance is a human monoclonalantibody.

The term “functional fragment” in the sense of the invention describes apolypeptide that has at least one of the biological activities that isalso shown by the entire polypeptide. In the case of antibodies, it isknown, for example, that not all CDR regions are necessary for thespecific binding. That is to say that the specific binding of theantibody can be effected by only one CD region, although a total of 3 CDregions are present. The specific binding of the antibody to an antigencan lead, for example, to the induction of apoptosis or to theinhibition of cell proliferation. The biological activity of afunctional fragment can be measured by various methods known to theperson skilled in the art. A method for measuring the interactionbetween antibodies and LDL, in particular LDL cholesterol, is the ELISAmethod.

The complementarity-determining regions (CDRs) of the polypeptidesequence includes the amino acid sequence that is essentially identicalto the amino acid sequence Ser-Gly-Asp-Lys-Leu-Gly-Asp-Lys-Tyr-Ala-Cys(CDR1), amino acids 23-33 of SEQ ID NO:1, Gln-Asp-Ser-Lys-Arg-Pro-Ser(CDR2), amino acids 49-55 of SEQ ID NO:1, andGln-Ala-Trp-Asp-Ser-Ser-Ile-Val-Val (CDR3), amino acids 88-96of SEQ IDNO:1 of the variable region of the light chain (VL); see also Page 2 ofthe Listing.

The complementarity-determining regions (CDRs) of the peptide sequenceinclude amino acid sequences that are essentially identical toSer-Tyr-Ala-Met-His (CDR1),Val-Ile-Ser-Tyr-Asp-Gly-Ser-Asn-Lys-Tyr-Tyr-Ala-Asp-Ser-Val-Lys-Gly(CDR2) and Asp-Arg-Leu-Ala-Val-Ala-Gly-Arg-Pro-Phe-Asp-Tyr (CDR3) of SEQID No 3 of the variable region of the heavy chain (V_(H)); see also Page4 of the Listing.

A polypeptide or a nucleic acid sequence is termed “essentiallyidentical” if it has at least 75%, 80%, 85% or 90% [identity] with theamino acid sequence (SEQ ID NO: 1 and 3) quoted as reference or with thenucleic acid sequence (SEQ ID NO: 2 and 4). In a further development ofthe polypeptide or of the nucleic acid sequence, at least 95%, 98%, 99%or 100% identity in comparison to the quoted references can bedemonstrated. For polypeptides, the length of the comparison sectionwill in general have at least 5, 10,15 or desirably at least 20 or 25sequential amino acids.

The polypeptide according to the invention can be generated by a methodknown by the name hybridoma technology (Köhler, Millstein, Nature, 1975,Vol. 256 495) and permits the isolation of monoclonal antibodies. It isbased on the in-vitro isolation of cellular hybrids that are obtained bythe cell fusion of normal lymphocytes with myeloma cells (e.g. HAB-1) ofunlimited viability and reproducibility. The hybridoma cells produced inthis way have the properties of both parent cells. Correspondingly theyhave the capability of lymphocytes to produce antibodies (e.g. SAM 6.10)and the capability of myeloma cells for unlimited division and therebyto produce antibodies in large amounts. Each hybrid cell resulting fromthe fusion produces monoclonal antibodies whose specificity isdetermined by the original lymphocyte cell. The hybridoma cells arereproduced and then those selected that produce antibodies of thedesired specificity. The cultivation of this selection and theirisolation leads to antibodies with highly specific reactivity, whichonly react with particular antigenic determinants.

The evidence of the substantial reduction of the low density lipoprotein(LDL) level (or of the LDL cholesterol level) or of the respectiveoxidized form in the blood plasma has been confirmed in an animalexperiment without the HDL level in the detectable range falling. Anessential feature of these is that the vital functions of the animalsare not affected during administration of the antibody, so that thesubstance according to the invention can so far be described as free ofside effects. (The mechanism of the antibody according to the inventioncould be explained by analogy with the mechanism of the known scavengerpathway.)

Another indication for the use of the medicament according to theinvention is the treatment of renal diseases, in particularglomerulonecrosis (glomerulosclerosis).

It lies within the scope of the invention that the polypeptide accordingto the invention is preferably used in purified form for the preparationof the drug, all methods known to a person skilled in the art (e.g.affinity chromatography, gel filtration) coming into consideration forpurification. As indication of the substance according to the invention,the fat-reducing effect is foremost, with emphasis on the selectivereduction of LDL or LDL-cholesterol. As a result of the property ofbinding LDL and/or oxLDL more strongly than HDL, the polypeptideaccording to the invention leads to a low value for the quotients ofLDL/HDL and/or oxLDL/HDL, and thus minimizes the infarct risk.

The adjuvants and carrier substances for preparing a drug are known to aperson skilled in the art and can be produced by conventional means (seeRemington: The Science and Practice of Pharmacy (20th ed.), ed. A. R.Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia ofPharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan,1988-1999, Marcel Dekker, New York).

Material and Methods

Immortalization of Lymphocytes and Primary Testing of the Antibodies

The lymphocytes are immortalized by fusing with the heteromyeloma HAB-1(Faller et. al, 1990) according to the standard protocol and incubating.In summary, lymphocytes are fused with HAB-1 cells by means of PEG. Thetriomes are seeded on four 24-well plates. The average growth frequencyis 80-90%; 50% of the growing clones secrete immunoglobulins. The firsttesting of the secreted human monoclonal antibodies was carried out byELISA to determine the isotype. The human monoclonal antibodies cansubsequently by analyzed by immunohistochemical, genetic, biochemicaland molecular biological techniques.

Required Materials:

-   -   RPMI 1640 (PAA company) without supplements RPMI 1640 with HAT        supplement (HAT-Supplement, PAA company) and 10% FCS, 1%        glutamine and 1% penicillin/streptomycin    -   Wash HAB-1 (fusion partner) twice with RPMI without supplements    -   Centrifuge for 5 min. at 1,500 rpm    -   thaw out frozen lymphocytes (from spleen, lymph nodes or blood)        and wash twice with RPMI without supplements; also centrifuge.    -   Suspend the two pellets in 10 ml RPMI without supplement each,        and count in the Neubauer counting chamber    -   Fuse Hab-1 to lymphocytes in a ratio of 1:2-1:3    -   Bring the cell pellets together after the second washing, mix        and centrifuge for 8 min. at 1,500 rpm    -   Carefully drip the PEG (Polyethylene Glycol 1500, from Roche),        which was previously heated to 37° C., onto the pellet with        gentle rotary movement of the 50 ml test tube    -   Resuspend slightly and then allow to rotate for exactly 90 s in        the water bath at 37° C.    -   Then wash out the PEG with RPMI without supplements (two full 10        ml pipettes)    -   Centrifuge for 5 min. at 1,500 rpm    -   Plate-out the 24-well plates with 1 ml per well of RPMI with HAT        supplement (HAT=hypoxanthine, aminopterine, thymidine)    -   Dissolve the pellet in RPMI with HAT supplement    -   Pipette one half-ml of the cells in each case into a 24-well        plate    -   Place the fusion plates in the incubator    -   Exchange the medium with RPMI with HAT supplement once per week

Purification of the SAM 6.10 Antibody

Purification of the culture supernatant by cation-exchangechromatography via FPLC

For this purpose, the hybridoma cells producing the SAM-6.10 lgMantibodies (DSM ACC2903, deposited Apr. 3, 2008, DSMZ, Inhoffenstr. 7BD-38124 Braunschweig) were cultured in a special serum-free cell culturemedium (AIMV medium, Gibco) and the lgM content in the culturesupernatant was determined by nephelometry. For purification, theculture supernatant was adjusted to a pH of 5.9 and the solutionfiltered. For binding, a special cationic column (HiTrap™ SP FF column,5 ml, Amersham Bioscience) was used. The column was equilibrated withfiltered buffer A (20 mM phosphate buffer, pH 5.9) at the beginning ofpurification. Then the culture supernatant cooled on ice was applied tothe column at a flow rate of 1 ml/min. After application of thesupernatant, the column was washed with buffer A for 20 min at a flowrate of 2 ml/min, until a constant base line so that not all boundproteins are removed. Subsequently the antibodies bound to the columnwere eluted by admixing buffer B (20 mM phosphate buffer, 1 M NaCl, pH8.0) and collected in fractions. The SAM-6.10 antibody (lgM) content inthe individual fractions was subsequently determined by nephelometry andthe purity and intactness of the purified antibody was tested bySDS-PAGE and western blot analysis.

Measurement of oxLDL

Measurement is carried out by means of the Oxidized LDL ELISA test kitfrom Mercodia, Uppsala, Sweden. Test principle: Oxidized LDL ELISA is asolid phase two-site enzyme immunoassay. It is based on the directsandwich technique in which two monoclonal antibodies are directedagainst a specific antigen on oxidized LDL apolipoprotein B. Duringincubation, oxidized LDL in the sample reacts with anti-oxidized LDLantibodies bound to wells in the microtitre plate. After washing, inwhich the non-reactive components of the sample are removed, aperoxidase-conjugated anti-human apolipoprotein B antibody detects theoxidized LDL that is bound to the solid phase. After a second incubationand washing, which removes the antibodies marked with the unboundenzyme, the conjugate is detected by reaction with 3, 3′, 5,5′tetramethylbenzidine (TMB). The reaction is stopped by the addition ofacid to define a calorimetric end point, which is readspectrophotometrically at 450 nm.

Measurement of the Binding of SAM-6 to oxLDL

An ELISA plate (Becton Dickinson Labare Europ, France) was incubatedovernight at 4° C. with the LDL fractions oxidized to different extents.The non-specific binding sites were blocked with RPMI 1640 mediumcontaining 10% FCS for 1 h. Then the plate was incubated with 60 μg/mlSAM-6 antibody for one hour at 37° C. After washing three times withPBS, the ELISA plate was incubated for 1 h with HRP-coupled secondaryantibody (rabbit anti human lgM, Dako Hamburg, Germany, 1:1000 in PBS).Then the plate was washed again with PBS and citrate buffer, OPD(DakoCytomation, Glostrup, Denmark) added and the colour change at 490nm was measured with an ELISA reader.

Flow Cytometry (FACS Analysis)

The adherent cells used were detached from the base of the culturebottles by treatment with trypsin/EDTA) The reaction was stopped with 10ml RPMI 1640 medium (+supplements) and the cells were pelleted at 1000×gfor 5 min. The cells were washed twice with PBS, suspended in FACSbuffer (PBS, 0.01% Na azide) and put on ice for 30 min to reconstitutethe cell membranes. Then the cells were adjusted to a density of 1×10⁶cells/ml and 200 μl of the cell suspensions were transferred to the FACSreaction vessels so that there was a cell count of 2×10⁵ cells per testtube. The cells were pelleted for 5 min. at 4° C. and 1,400 rpm, thepellets resuspended and incubated with the primary antibodies for 15min. on ice. As primary antibodies, 100 μg/ml SAM-6 antibodies in FACSbuffer (total volume 200 μl) or 100 mg/ml LDL were used. 100 μg/mlChrompure human lgM acted as isotype control. Alternatively, the cellswere preincubated with LDL for 30 min. and then 100 μg/ml SAM-6antibodies added for 15 min.

After incubation, the cells were centrifuged off; the supernatant wasdiscarded and the pellets were washed with 500 μl cold FACS buffer. Thiswas followed by a 15-minute light-protected incubation with theFITC-coupled secondary antibody (rabbit anti human lgM, FITC-coupled,1:50 in FACS buffer for SAM-6 or Chrompure human lgM). After washingagain, the cells were suspended in 200 μl cold FACS buffer and stored,light-protected, on ice until measurement. Measurement was carried outby means of a flow cytometer (FACScan; Becton Dickinson, USA).

Animal Experiments for Demonstrating the in vivo Effect of the SAM 6.10Antibody

Experiment 1: 500 μg purified SAM 6.10 antibodies was injectedintraperitoneally. The LDL concentration in the blood serum was measuredafter 2 days (see below for method).

Experiment 2: as above

Experiment 3: 1 mg purified SAM 6.10 antibodies was injectedintraperitoneally. The LDL concentration in the blood serum was measuredafter 14 days.

-   -   Control A: Normal values control mouse    -   Control B: Normal values control mouse

The serum concentration of LDL is significantly reduced in the micetreated with SAM 6.10 (see FIGS. 10 and 11).

Toxicity

500 μg and 1 mg respectively of purified SAM 6.10 antibodies wasinjected intraperitoneally into mice.

-   -   no acute toxicity    -   no latent toxicity (period 3 months).

The organs of the dead mice from experiments 1, 2 and 3 (see above) wereremoved and investigated: The liver, lung, heart, spleen, smallintestine, large intestine, kidneys, stomach and brain did not show anymorphological changes. The organs were furthermore investigatedimmunohistochemically for any lipid deposits. Staining with Sudan IIIdid not show lipid deposits in any organ.

The organs of the sacrificed mice were fixed in formalin and embedded inparaffin. Staining was carried out with Sudan III dye according to thefollowing protocol:

Sudan III Staining on Paraffin Sections/for Macrophages

Deparaffining:

-   -   Xylene 1 5 min.    -   Xylene 2 5 min.    -   100% Ethanol 1 5 min    -   100% Ethanol 2 5 min    -   Methanol 70 ml +H₂ 0 ₂ 500 μl 5 min    -   90% Ethanol 1 3 min    -   90% Ethanol 2 3 min    -   80% Ethanol 1 3 min    -   80% Ethanol 2 3 min    -   70% Ethanol 1 3 min    -   70% Ethanol 2 3 min    -   Place sections in PBS    -   Incubate sections for 15 min with Sudan III    -   Wash with aqua dest.    -   Immerse 1× in 60% isopropanol    -   Wash with aqua dest.    -   Counter stain with haemalaun for 6 min.    -   Soak sections for 10 min, wash with aqua dest and mount in        glycerine gelatine

For Sudan III staining of macrophages, the adherent macrophages aregrown on object slides and then treated with the corresponding reagents.Staining is carried out as follows:

-   -   Fix the cells in 60% isopropanol (6 min)    -   Incubate for 20 min with Sudan III    -   Wash with aqua dest.    -   Counterstain with haemalaun for 6 min    -   Soak sections for 10 min, wash with aqua dest and mount in        glycerine gelatine

Determination of Lipids in Blood Samples

Measurement of the various lipids in blood serum was carried outautomatically with the MODULAR D P800 instrument (Roche). Determinationof the LDL cholesterol value was carried out by enzymatic colorimetry(CHOD/PAP) without sample pre-treatment.

Test Principle for Determining the Lipid Concentration in Serum:

HDL, VLDL and chylomicrons are selectively hydrolysed by a detergent 1.The cholesterol liberated in these lipoproteins reacts immediately as aresult of the enzymatic action of the cholesterolesterase (CE) andcholesteroloxidase (CHOD), and hydrogen peroxide is produced. The latterforms a colourless product with 4-aminoantipyridine in the presence of aperoxidase (POD). During this step, the LDL particles remain intact. Thereaction of LDL cholesterol is initiated by the addition of detergent 2and the coupling substance N,N-bis(4-sulphobutyl)-m-toluidine (DSBmT).The second detergent liberates cholesterol in the LDL particles. in theenzymatic reaction, a dye is formed in the presence of the couplingsubstance. The intensity of the red quinine imine dye formed is directlyproportional to the LDL cholesterol concentration. It is determined bymeasuring the increase of extinction at 552 nm.

ELISA (LDL/HDL)

-   -   Precoat the ELISA plate with LDL (Lipoprotein low density from        Human Plasma, Sigma, 10 μg/ml in PBS) or HDL (human HDL,        Chemicon, 10 μg/ml in PBS)→50 μl per well    -   Cover plate and store overnight at 4° C.    -   Next day, wash plate 2× with PBS    -   Pipette 100 nl of RPMI into each well and incubate for 1 h at        room temperature    -   Then wash 2× with PBS    -   Pipette 50 μl in each case of the respective positive controls        into 2 wells (double determination) Positive control: Mab to        human LDL mouse IgG2a 1:1000 in PBS    -   Alongside, 50 μl RPMI as negative control (double determination)    -   Pipette 50 μl of the samples (supernatant SAM6.10) (double        determination) side by side    -   Incubate for 1 h in the incubator    -   Wash 2× with PBS    -   Wash 2× with PBS/0.05% Tween    -   Wash 2× with PBS    -   Pipette 50 μl of the respective second AK (peroxidase        conjugated): Rabbit anti human lgM 1:1000 in PBS/0.05% Tween        (for SAM 6.10) Rabbit anti mouse IgGs 1:1000 with PBS Tween (for        positive control LDL)    -   Incubate for 1 h in the incubator    -   Wash 2× with PBS    -   Wash 1× with PBS/0.05% Tween    -   Wash 2× with PBS    -   Wash 2× with citrate buffer    -   For evaluation: Dissolve OPD tablet (Dako, Hamburg) in citrate        buffer+H₂O₂ (3 ml citrate buffer+one tablet+5 μl H₂O₂)    -   Pipette 50 μl dye into each well    -   When there is a positive reaction yellow coloration), stop with        10 μl 3 M H₂SO₄

Sequence listing

Page 1of the Listing shows the amino acid sequences (SEQ ID NO:1) of thevariable region of the light chain (V_(L)).

Page 2of the Listing shows the nucleotide acid sequences (SEQ ID NO:2)of the variable region of the light chain (V_(L)). Thecomplementarity-determining regions (CDRs) are indicated by horizontallines and are substantially identical to nucleotides 67-99 (CDR1),145-165 (CDR2) and 262-288 (CDR3) of SEQ ID NO:2.

Page 3of the Listing shows the amino acid sequences (SEQ ID NO:3) of thevariable region of the heavy chain (V_(H)).

Page 4 of the Listing shows the nucleotide acid sequence (SEQ ID NO:4)of the variable region of the heavy chain (V_(H)). Thecomplementarity-determining regions (CDRs) are indicated by horizontallines and are substantially identical to nucleotides 91-105 (CDR1),148-198 (CDR2) and 295-330 (CDR3) of SEQ ID NO:4.

Cell-biological experiments

DESCRIPTION OF FIGURES

The figures explained below are not intended to restrict the invention,but only to explain it and prove its feasibility with reference toexamples.

FIG. 1 shows the measurement of oxLDL in dependence on the incubationtime with a copper sulphate solution. In the experiment, LDL (Sigma,Taufkirchen, Germany) was oxidized for 3 and 15 h respectively byincubation with 20 μM CuSO₄. The amount of oxidized LDL was determinedwith the Mercodia Oxidized LDL ELISA, according to the instructions foruse. It can be clearly seen that the amount of oxidized LDL increaseswith increasing incubation time, with each LDL fraction that was nottreated with copper ions being already partly present in oxidized form.After 15 hours' incubation, the proportion of oxidized LDL hasapproximately doubled.

FIG. 2 shows the proof of binding of SAM-6 to oxLDL. To prove binding ofSAM-6 to oxLDL by means of the ELISA binding assay, the ELISA plate wasprecoated with LDL fractions oxidized to different degrees before theprimary antibody SAM-6 and the secondary antibody anti-human lgM,required for detection purposes, were added. The result shows that themore LDL that is present in its oxidized form, the more strongly theantibody SAM-6 according to the invention binds.

FIG. 3 shows the result of an FACS analysis. The cells used for thispurpose are of the mouse macrophage cell line P388D1(lL-1) (DSMZAccession No. ACC 288). Figure 3A shows the binding of LDL tomacrophages. Figure 3B proves that the human monoclonal antibody SAM-6,too, binds to macrophages. The proof of binding of a control lgM tomacrophages in Figure 3C demonstrates that the macrophages possess μreceptors. The rightward shift of the signal in Figure 3D demonstratesthat simultaneous incubation of LDL and SAM-6 effect a multiple bindingof SAM-6 to the cells.

FIGS. 4 and 5 show the result of the Sudan III staining, for which thecells of the mouse macrophage cell line P388D1(lL-1) were used. Figure gFIG. 4 shows cells that were incubated for 48 h with either SAM-6 or anlgM control antibody and subsequently subjected to Sudan III staining.The cells incubated with the antibody SAM-6 show, by their redcoloration, distinct deposition of neutral fats. The cells incubatedwith the control antibody by contrast show no changes.

For the stains shown in FIG. 5, the macrophages were cultivated for 24 hboth with and without FCS supplement. Then, for a further 24 h, eitheronly LDL, or only SAM-6, or LDL and SAM-6 together were respectivelyadded. Subsequently staining with Sudan III was carried out. The leftfigure column with the FIGS. 5A, 5C and 5E show cells that werecultivated without supplementation of FCS. The right image column withthe FIGS. 5B, 5D and 5F show cells that were cultivated withsupplementation of FCS.

FIGS. 5A and 5B demonstrate that both macrophages that were cultivatedwithout FCS and macrophages that have grown in the presence of FCS showbasal deposition of neutral fats. Figure 5C demonstrates that when SAM-6is supplemented to macrophages that were cultivated without FCS, no fatdeposition is evident. As shown in Figure 5D, on the other hand, areinforced lipid accumulation is observed in macrophages that werecultivated with FCS and subsequently had SAM-6 added to them.

FIGS. 5E and 5F demonstrate that, with a co-incubation of SAM-6 and LDL,the intracellular lipid accumulation increases substantially both inmacrophages that were cultivated with FCS and in those that have grownwithout the addition of FCS.

FIG. 6 shows the effect of the antibody SAM-6 on LDL values in vivo. Inthe experiment, 1 mg of purified SAM-6 antibodies and, in the controlexperiment, 1 mg of human Chrompure lgM (isotype control) were injectedinto the mice intraperitoneally. The LDL concentration in the blood wasmeasured after 24 and 48 h. After 24 and 48 hours, a clear reduction ofserum LDL can be observed in the mice treated with SAM-6.

The measurement of LDL in the blood serum was carried out automaticallywith the MODULAR D P800 instrument (Roche), the plotted values beingobtained as a result of the “LDL Cholesterol Direct” diagnostic kit(Roche Diagnostics).

FIG. 7 also demonstrates the in vivo effect of SAM-6.10, an indirectmethod according to the Friedewald formula being used to calculate thevalues. Here, the amount of LDL is calculated from the differencebetween total cholesterol, HDL and triglycerides. According to thismanner of evaluating the experiment, the reduction of the concentrationof LDL in the serum after SAM 6.10 treatment is even greater. However,this indirect measurement method must be regarded as less accurate incomparison to the method used in FIG. 6.

The invention claimed is:
 1. A purified antibody or functional fragmentthereof, comprising a light chain (V_(L)) variable region sequence and aheavy chain (V_(H)) variable region sequence: wherein the heavy chain(V_(H)) variable region sequence comprises an amino acid sequence atleast 90% identical to the amino acid sequence of SEQ ID NO.:3, whereinsaid antibody or functional fragment thereof binds to apolipoprotein Bcontaining low density lipoproteins (LDL) and apolipoprotein Bcontaining oxidized LDL (oxLDL).
 2. The purified antibody or functionalfragment thereof according to claim 1, wherein said antibody orfunctional fragment thereof binds to apolipoprotein B containing LDLcholesterol and binds to apolipoprotein B containing oxidized LDLcholesterol (oxLDL cholesterol).
 3. The purified antibody or functionalfragment thereof according to claim 1, wherein said apolipoprotein Bcontaining low density lipoproteins (LDL) or said apolipoprotein Bcontaining oxidized LDL (oxLDL) occur in humans.
 4. The purifiedantibody or functional fragment thereof according to claim 1, whereinsaid antibody or functional fragment thereof is a functional fragment ofsaid antibody.
 5. The purified antibody or functional fragment thereofaccording to claim 1, wherein said functional fragment is selected fromthe group consisting of F_(v), Fab, Fab′ and F(ab′)₂.
 6. The purifiedantibody or functional fragment thereof according to claim 1, whereinthe light chain (V_(L)) variable region sequence is at least 80%identical to SEQ ID NO:1.
 7. The purified antibody or functionalfragment thereof according to claim 5, wherein said functional fragmentcontains an amino acid fragment of the light chain (V_(L)) variableregion or the heavy chain (V_(H)) variable region amino acid sequence ofSEQ ID NO:1 or SEQ ID NO:3.
 8. The purified antibody or functionalfragment thereof according to claim 1, wherein said light chain (V_(L))variable region sequence is at least 85% identical to the amino acidsequence of SEQ ID NO:1.
 9. The purified antibody or functional fragmentthereof according to claim 1, wherein said heavy chain (V_(H)) variableregion sequence is at least 95% identical to the amino acid sequence ofSEQ ID NO:3.
 10. The purified antibody or functional fragment thereofaccording to claim 1, wherein said light chain (V_(L)) variable regionsequence is at least 90% identical to SEQ ID NO:1.
 11. The purifiedantibody or functional fragment thereof according to claim 1, whereinsaid heavy chain (V_(H)) variable region sequence is at least 98%identical to SEQ ID NO:3.
 12. The purified antibody or functionalfragment thereof according to claim 1, wherein said antibody orfunctional fragment thereof is a monoclonal antibody.
 13. The purifiedantibody or functional fragment thereof according to claim 1, whereinsaid antibody or functional fragment thereof is produced by a hybridoma.14. The purified antibody or functional fragment thereof according toclaim 1, wherein said light chain (V_(L)) variable region sequence ofthe antibody or functional fragment contains a sequence at least 95%identical to SEQ ID NO:1.
 15. The purified antibody or functionalfragment thereof according to claim 1, wherein said light chain (V_(L))variable region sequence of the antibody or functional fragment containsa sequence at least 98% identical to SEQ ID NO:1.
 16. The purifiedantibody or functional fragment thereof according to claim 1, whereinsaid heavy chain (V_(H)) variable region sequence of the antibody orfunctional fragment contains a sequence at least 99% identical to SEQ IDNO:3.
 17. A purified antibody or functional fragment thereof comprisinga light chain (V_(L)) variable region sequence and a heavy chain (V_(H))variable region sequence, wherein the light chain (V_(L)) and heavychain variable region sequences comprise SEQ ID NO:1 or SEQ ID NO:3,respectively, and wherein said antibody or functional fragment thereofbinds to apolipoprotein B containing low density lipoproteins (LDL) andapolipoprotein B containing oxidized LDL (oxLDL).
 18. A purifiedantibody or functional fragment thereof, wherein the heavy chain (V_(H))variable region sequence comprises a complementary-determining region(CDR) set forth as [Ser-Tyr-Ala-Met-His (CDR1) amino acids 31-35 of SEQID NO:3, and Val-Ile-Ser-Tyr-Asp-Gly-Ser-Asn-Lys-Tyr-Tyr-Ala-Asp-Ser-Val-Lys-Gly (CDR2) amino acids 50-66 of SEQ ID NO:3, andAsp-Arg-Leu-Ala-Val-Ala-Gly-Arg-Pro-Phe-Asp-Tyr (CDR3) amino acids99-110 of SEQ ID NO:3.], and wherein said antibody or functionalfragment thereof binds to apolipoprotein B containing low densitylipoproteins (LDL) and apolipoprotein B containing oxidized LDL (oxLDL).19. The purified antibody or functional fragment thereof according toclaim 18, wherein said heavy chain (V_(H)) variable region sequence ofthe antibody or functional fragment contains a sequence at least 90%identical to SEQ ID NO:3.
 20. The purified antibody or functionalfragment thereof according to claim 18, wherein the light chain (V_(L))variable region sequence comprisesSer-Gly-Asp-Lys-Leu-Gly-Asp-Lys-Tyr-Ala-Cys (CDRI), amino acids 23-33 ofSEQ ID NO:1, Gln-Asp-Ser-Lys-Arg-Pro-Ser (CDR2), amino acids 49-55 ofSEQ ID NO:1, or Gln-Ala-Trp-Asp-Ser-Ser-Ile-Val-Val (CDR3), amino acids88-96 of SEQ ID NO:1.
 21. The purified antibody or functional fragmentthereof according to claim 18, wherein the light chain (V_(L)) variableregion sequence comprises Ser-Gly-Asp-Lys-Leu-Gly-Asp-Lys-Tyr-Ala-Cys(CDR1), amino acids 23-33 of SEQ ID NO:1, Gln-Asp-Ser-Lys-Arg-Pro-Ser(CDR2), amino acids 49-55 of SEQ ID NO:1, andGln-Ala-Trp-Asp-Ser-Ser-Ile-Val-Val (CDR3), amino acids 88-96 of SEQ IDNO:1.
 22. A purified antibody or functional fragment thereof, whereinthe heavy chain (V_(H)) variable region sequence comprisesSer-Tyr-Ala-Met-His (CDR1), amino acids 31-35 of SEQ ID NO:3,Val-Ile-Ser-Tyr-Asp-Gly-Ser-Asn-Lys-Tyr-Tyr-Ala-Asp-Ser-Val-Lys-Gly(CDR2), amino acids 50-66 of SEQ ID NO:3, andAsp-Arg-Leu-Ala-Val-Ala-Gly-Arg-Pro-Phe-Asp-Tyr (CDR3), amino acids99-110 of SEQ ID NO:3, and wherein said antibody or functional fragmentthereof binds to apolipoprotein B containing low density lipoproteins(LDL) and apolipoprotein B containing oxidized LDL (oxLDL).
 23. Apurified polypeptide, comprising a heavy chain (V_(H)) variable regionsequence at least 90% identical to the amino acid sequence of SEQ IDNO.:3, wherein said polypeptide binds to apolipoprotein B containing lowdensity lipoproteins (LDL) and apolipoprotein B containing oxidized LDL(oxLDL).
 24. The purified polypeptide of claim 23, wherein the heavychain (V_(H)) variable region sequence is at least 95% identical to theamino acid sequence of SEQ ID NO.:3, and wherein said polypeptide bindsto apolipoprotein B containing low density lipoproteins (LDL) andapolipoprotein B containing oxidized LDL (oxLDL).
 25. The purifiedpolypeptide of claim 23, wherein the heavy chain (V_(H)) variable regionsequence is at least 98% identical to the amino acid sequence of SEQ IDNO.:3, and wherein said polypeptide binds to apolipoprotein B containinglow density lipoproteins (LDL) and apolipoprotein B containing oxidizedLDL (oxLDL).
 26. The purified polypeptide of claim 23, wherein the heavychain (V_(H)) variable region sequence is at least 99% identical to theamino acid sequence of SEQ ID NO.:3, and wherein said polypeptide bindsto apolipoprotein B containing low density lipoproteins (LDL) andapolipoprotein B containing oxidized LDL (oxLDL).
 27. The purifiedpolypeptide of claim 23, wherein the heavy chain (V_(H)) variable regionsequence comprises [Ser-Tyr-Ala-Met-His (CDR1) amino acids 31-35 of SEQID NO:3, andVal-Ile-Ser-Tyr-Asp-Gly-Ser-Asn-Lys-Tyr-Tyr-Ala-Asp-Ser-Val-Lys-Gly(CDR2) amino acids 50-66 of SEQ ID NO:3, andAsp-Arg-Leu-Ala-Val-Ala-Gly-Arg-Pro-Phe-Asp-Tyr (CDR3) amino acids99-110 of SEQ ID NO:3.